JPH06340699A - Antigen concerning human nonmicrocell lung cancer - Google Patents

Abstract

PURPOSE: To provide an antigen compsn. purified from a culture medium of human non-small cell lung carcinoma contg. a specified protein, excellent in monoclonal antibody production ability useful as a diagnostic marker.

CONSTITUTION: An objective antigen compsn. is purified from a culture medium for human non-small cell lung carcinoma contg. L3 protein which has a terminal amino acid sequence of the formula in an unmodified and glycosylated form I, which has a molecular weight of about 94000 Daltons determined by SDS polyacrylamide gel electrophoresis, and which has an N-bonded glycoside residue wherein the terminal residue is sialic acid residue.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

This invention relates to human non-small cell lung cancer (NSC).
LC) novel antigen composition.

[0002]

Background 1 Background of the Invention invention. FIELD OF THE INVENTION Human lung cancer is responsible for many deaths from carcinomas in men and the process of breast cancer invasion as the most frequent cause of carcinoma deaths in women (Cancer).
Facts and Figers, 1983 [Cancer Facts
and Figures, 1983]). This disease has four major histological types: epidermoid (30).
%), Adenocarcinoma (35%), undifferentiated large cells (15%) and small cells (20%)
%). Many cases of lung cancer are incurable by chemotherapy and radiation therapy. Small cell lung cancer responds to chemotherapy and radiation therapy by a reduction in size, but not overall cure. Complete surgical removal of the tumor is considered the only effective therapy. However, unexpectedly, less than 30% of lung cancer patients have tumors that can be completely resected diagnostically, and less than 1/3 of lung cancer patients have five years after complete surgical removal. Alive Therefore, there is a great need for methods that allow for earlier diagnosis of lung cancer, better definition of the extent of cancer spread, and more effective therapy.

Monoclonal antibodies can be used for all these purposes. However,
A prerequisite is to find antibodies to the antigen that are more strongly expressed in lung cancer than in normal adult tissues. Known heterogeneity of tumor cell populations, presence of several determinants on the same antigen, expected differences between antibodies regarding their suitability as diagnostic markers when compared to therapeutic targets, and the same antigen In view of the different biological properties of the different antibodies against, several different antibodies are required. 2. Description of the Prior Art Monoclonal antibodies against lung cancer antigens have been described by Shikora et al. [Sik
ora et al.] (British Journal of Cancer (1981) [Br.J.Cancer (1981)] Volume 43, 696-700
Page), and Cuttatta et al. [Process of National Academy Science United States of America (1981) [Proc.Natl.Acad.Sci.USA (1981)] Vol. 78.
4591-4595) and Birki et al. [Varki et al.] (Cancer Research (1984) [Cancer Research (1984)] 44th.
Volume 3681-687) and tea. W. Mandy et al. [Mondy, TW et al.] (Science (1981) [Scie
nce (1981)] Vol. 214, pp. 1246-1248), and J. Dee. Mina, JD et al. (In Vitro [In Vi
tro] 17 (12) 1058-1070 (12-1981)) and A. J. [Kennel, AJ et al.] (Cancer Research) 41 (9, PT1) 3465-3470
Page, Chemical Abstracts [Chem.Abst.] No. 95 (15) No.
1308502), ray. Bee. Baylin, ABet a
l.] (Process of National Academy Science USA [Proc.Natl.Acad.Sci.USA] Vol. 79
4650-4654 (8-1982)) and D. N. Carney, DNet al.] (Pasobiology Annual
[Pathobiology Annual] 1982 12th page 115-136 (19
1982)), A. F. Gazdar, AFet a
l.] (Seminars in Oncology
ology] Vol. 10, pp. 3-19 (3-1983)), A. C.
Hollinshead, AC. et al.] (Cancer Detec.Preven
t.] Vol. 6, pp. 185-191 (1983)), and Jay. tea. [Mulshine, JT et al.] (J. Immunol.] 131 (1) 497-502 (1983
Year)), Elle. C. Huang, LC et al.
(Architecture of Biochemistry and Biophysics [Arch.Bioch.Biophys.] No. 220
(1) Pages 318-320 (1983)), S. Surge et al. [Saj
i, S. et al.] (Hybridoma 3 (2) pp. 119-130 (19
1984), Bio Abstract [No. 790055]
69), Kay. Bosslet, K. et al.] (Behring Inst. Mitt.) Vol. 74
Pages 27-34 (1984), Chemistry Abstract [C
hem.Abst.] CA101 (9) No. 706686) and ray. tea. [Rosen, AT et al.] (Cancer Research [Can
cer Research] 44 (5) 2052-2061 (1984), Bio-Abstract [Bio. Abst.] No. 79014658), Gee. N. Pee. Van Muijen, G.
NPet al.] (Amer J. Patho
l.] 116 (3) pp. 363-369 (1984), Bioabst. [79023605], and Gee. Jay. Princler, GJ et al. (Cancer Research, Vol. 42, pp. 843-848 (3-1982)) and tea. Mazauric, T.
et al.] (Cancer Research] 42nd
Vol. 150-154 (1-1982)) and Jay. A. Braats, JA et al. (Cancer Research
[Cancer Research] Vol. 42, pp. 849-855 (3-1982))
Or Earl. E. Sobol et al. [Fed. Proc.] 41 (3) 409, Abstract [Abst.] 816 (4-1982).

[0004] Continuous culture of fused cells secreting antibodies of a predetermined specificity has been described by Kohler et al.
l.] (Nature (1975) 265: 495 ~ 497 [Nature (1975)
265: 495-497]). Means for producing tumor antibodies are described in US Pat. No. 4,172,124.

[0005]

The present invention relates to a novel antigen composition for human non-small cell lung cancer (NSCLC). "NSCLC
The term "cell" is meant to include epidermoid carcinoma cells, adenocarcinoma cells and anaplastic large cell carcinoma cells. The determinant site is also an antigen of some other cancer, eg some cancers of the breast. And thus the antibodies of the invention also bind to these other cancer cells.These monoclonal antibodies bind to normal adult cells to a lesser extent than to tumor cells. The term "to a lesser extent" means that the binding may not be detectable by immunohistological techniques, or that NSCLC cells have binding as determined by immunohistological techniques. It will be about 4 to 5 times smaller than it binds to the monoclonal antibody, which is secreted by the mouse hybridoma.

The invention also relates to some diagnostic methods using the monoclonal antibodies according to the invention.
One such method involves measuring the presence of NSCLC cells in a specimen suspected of containing such cells. The sample is contacted with a monoclonal antibody, which is what distinguishes such cells from other cell types that may be present in the sample. Contacting is performed under conditions for the binding of antibody to such cells. After contacting, the presence or absence of binding of antibody to such cells in the analyte is measured. This binding is
It is related to the presence or absence of NSCLC cells.
Generally, the analyte is contacted with a labeled specific binding partner for the monoclonal antibody. This indicator is capable of emitting a detectable signal.

[0007]

The present invention is directed to antibodies specific for antigens on human NSCLC cells and functional equivalents, binding fragments and immunoconjugates of such antibodies, as well as the use of such antibodies. It relates to a diagnostic method. For example, the monoclonal antibodies of the invention can be produced by the standard techniques of Koehler and Milstein (supra). For example, human lung cancer cells from multiple exudates or cultured cells from human non-small cell lung cancer, or cells from normal fetal lung are used as immunogens. These cells are injected into mice and after a sufficient time the mice are sacrificed and splenocytes obtained. The splenocyte chromosome encoding for the desired immunoglobulin is immortalized by fusing the splenocytes with myeloma cells or lymphoma cells, usually in the presence of polyethylene glycol. The resulting cells containing the fused hybridomas are grown in a selective medium such as HAT medium, and viable cells are grown in such medium using limiting dilution conditions. The cells were grown in a suitable container, such as a microtiter well, and the supernatant was screened for monoclonal antibodies with the desired specificity.

Various techniques exist for increasing the yield of monoclonal antibodies, such as injecting hybridoma cells intraperitoneally into a mammalian host that receives the hybridoma cells and harvesting ascites fluid. If the monoclonal antibody is not collected in sufficient quantity in the ascites, it will be harvested from the blood of the host. Various well-known methods exist for the isolation and purification of monoclonal antibodies to free them from other proteins or other impurities (see Köhler and Milstein, supra).

One such monoclonal antibody of the present invention is exemplified by a novel monoclonal antibody called L3. This monoclonal antibody has a molecular weight of human NSCLC cells, that is, malignant cells, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
It defines determinant sites on cell-associated antigens with 76,000 properties and on protein antigens (forms I, II and III antigens) secreted by NSCLC cells into the culture medium. This antibody is of the IgG ₁ isotype. This L3 antibody is produced by an L3 mouse hybridoma.

Form I of L3 antigen is sodium dodecyl sulfate-polyacrylamide one-dimensional gel electrophoresis (SDS-PAG
In E), it has a molecular weight of 94,000. Form
The L3 antigen of II has a molecular weight of 76,000 in SDS-PAGE, and the L3 antigen of form III is SDS-PAGE.
It has a molecular weight of 26,000 in E. The Form I L3 antigen can be purified 9-fold from the culture medium by affinity chromatography (Table 1), and affinity chromatography showed that one of the glycoproteins, Form I L3 antigen, was 70% and 90%, respectively. This is followed by acrylamide gel technology to give compositions containing more than 100%. The composition resulting from the affinity chromatography purification has a specific activity of 46,500 units / mg versus 522 units / mg for the culture medium. This specific activity is defined as the amount of antigen required to block up to 50% binding of ¹²⁵ I-labeled L3 antibody in a competitive binding assay using formalin-immobilized monolayers of Calu-1 cells. It The composition obtained from the culture medium by affinity chromatography provided 70% of the Form I antigen.
% Or more and about 50,000 to 20 by SDS-PAGE
Non-specific protein material with a molecular weight between 0,000 3
Many parts less than 0% are lesser amounts of about 10
It contains 20% of Form II antigen. Minor amounts of Form III antigen are also present in the composition.

To determine the relationship between the cellular and extracellular forms of the L3 antigen, cells were radiolabeled with ³⁵ S methionine and pulsed chase experiments were performed. 1 cell
Pulsed with ³⁵ S methionine for different times, placed in medium containing unlabeled methionine for various times and subjected to immunoprecipitation analysis. First, all specific immunoprecipitable cell-bound radioactivity was found in the 76,000 protein.

The other labeled proteins observed were also found in the antibody-depleted samples. During the follow-up period, the radioactivity in the 76,000 MW form decreased while the radioactivity found in the form released into the medium (MW 94,000) increased. The radioactivity in the cell-bound form disappears with kinetics similar to that exhibited in the released form; t for both processes
_1/2 was determined to be about 1.5 hours by concentration quantitative analysis. These results show that the cell-bound molecular weight of the antigen
It is suggested that the 76,000 form is a precursor of the form having a molecular weight of 94,000 (Form I L3 antigen).

Because the L3 antigen is a glycoprotein, carbohydrate modification would be a conceivable explanation for the apparent increase in size of the L3 antigen observed during release from cells. This possibility was investigated by treating the immunoprecipitates containing the cellular and released forms of the molecule with glycosidases of known specificity. The size of the cell-bound L3 antigen was reduced to a molecular weight of 59,000 by treatment with endoglycosidase H (Endo H), but was unaffected by neuraminidase, which contains N-bridged high mannose oligosaccharides. (Talentino et al., 1974 J. Biol. Chem., 249: 811 ~ 817 [Tarentino, 1
974, J. Biol. Chem. 249: 811-817]). This result is consistent with the finding that maturation is sensitive to tunicamycin, a known inhibitor of N-bridge glycosylation (Refol et al., 1976 FB S Letters, 7).
1: 167〜170 [Lehle, et al., 1976, FEBS Letters, 71: 167-
170]). On the other hand, the released antigen is resistant to Endo H, but has a molecular weight of 80,0 by treatment with neuraminidase.
Reduced in size to 00, which is Form I of L3
It shows the presence of terminal sialic acid residues in the antigen. Therefore, conversion of the L3 antigen from the cell-bound form to the released form is achieved by maturation of the N-bridged carbohydrate side chains.

The amino acid terminal sequence of the Form I L3 antigen is
It was determined using well known techniques in compositions purified by a combination of affinity chromatography and acrylamide gel separation techniques. This array (α)
Was as follows: And further defined as:

[0015] The above-mentioned one-letter symbols for amino acids are V = valine, N = asparagine, D = aspartic acid, G = glycine, M = methionine, R = arginine, L = leucine, A = alanine, T = threonine, Q = glutamine, E. = Glutamic acid, I = isoleucine, F = phenylalanine, Y = tyrosine and W = tryptophan, with their well-known meanings. The above shows that regardless of the presence of the Form II L3 antigen, the Form I L3 antigen is present.
Obtained as a single sequence for compositions containing antigen.

The above 30 N-terminal amino acid sequences of the L3 antigen, the National Biochemical Research Foundation Protein Bank [the National
Biochemical Research Foundation protein bank] (1985
(March 2003) comparison with the existing protein sequences did not show any significant homology. The sequence of the Form I L3 antigen was also compared to that of several serum proteins not currently listed in this database, including human C1-esterase inhibitor and human α-2-thiol proteinase inhibitor. None of these sequences displayed any significant homology with the N-terminal sequence of the Form I L3 antigen.

We have determined that the Form I L3 antigen is present in normal human serum and that the Form I L3 antigen is purified from the serum. The properties of the L3 antigen from serum are summarized in Table 3. The Form I L3 antigen can be purified from normal human serum in 3000-fold or more with a yield of 39% (Table 2). Analysis of the purified material by SDS-PAGE showed that the major component was a protein that co-migrated with Form I and Form II components purified from the culture medium (neither form being a major component of the original serum sample). Revealed. It is noteworthy that both Form I and Form II L3 antigens from serum are split into two distinct components in the original gel, which is probably due to glycosylation differences. Shows microheterogeneity.
For both forms, following immunoblotting analysis,
It is also reactive after radioiodination and immunoprecipitation. In the case of the antigen purified from the culture medium, after immunoprecipitation, another form, the molecular weight of 26,000 (form III L
3 antigen) is detected. The composition obtained by immunoaffinity chromatography contains more than 50% by weight of Form I L3 antigen, the balance material is Form III L3 antigen, transferrin and SDS-PAGE with a non-specific molecular weight of about 50,000 to 200,000. It consists of a proteic protein. This composition has a specific activity of 45,500 units / mg versus 13.3 units / mg for normal human serum.

These results indicate that L3 antigenic activity is expressed in two components (form I and form II) found in both culture medium and serum. Further purification of the Form I L3 antigen from serum by suitable SDS-PAGE predominantly contained Form I L3 antigen at levels greater than 90% by weight, and indeterminate amounts of Form II.
It comprises antigen and transferrin and residual material which is a non-specific protein with a molecular weight of about 50,000 to 200,000.

Two-dimensional isoelectric focusing-SDS-PAGE
Purified from both serum and culture medium by ¹²⁵
The I-labeled and immunoprecipitated antigens were compared. The two-dimensional mobilities of both antigens are very similar, 4.2-5.0
I and Form II that migrate as heterogeneous species in the pH range of
Had ingredients. Form I L3 antigens from both sources also show two smaller components that migrate at estimated pH values of 5.8 and 6.2. The carbohydrate composition of both antigens was also examined by testing for glycosidase sensitivity. Form I and Form II L3 from both sources
The antigen was found to be EndoH resistant, whereas both neuraminidase treatments were found to result in the appearance of the characteristic 80,000 fragment found following treatment of the radiantly labeled antigen. The neuraminidase sensitivity of the Form II antigen indicates that it is different from the cell-bound form of the L3 antigen with a molecular weight of 76,000.

To confirm that both molecules are structurally similar, we described a Form I molecule from both forms by Kleberand et al. (1977) Jay. Biol. Chem. , 252: 1102-1106 [Cleveland et al., (1977), J. Bio.
l. Chem. 252: 1102-1106] and subjected to a series of partial protease cleavage reactions. Radiolabeled Form I antigen, purified from both culture medium and serum, was excised from preparative SDS-PAGE gels and fingerprinted with V8 protease. Both molecules produce the same partial cleavage pattern, at least 5
One restricted partial cleavage product is observed in both cases (Mw = 76,000, 49,000, 31,000, 20,000 and 14,300). These results indicate that the L3 antigen purified from culture medium and human serum is highly relevant.

We next investigated the relationship between the three forms of L3 antigen from serum. All fingerprints for Form I and Form II show that the Form II molecule is Form I.
It does not generate the molecular weight derived fragments of 31,000 and 14,300, but is very similar. This pattern implies that the Form I and Form II molecules are highly related. The Form III antigen shares a partial cleavage product of molecular weight 14,300 with the larger antigenic form found in serum,
This indicates that the Form III antigen is also structurally related to the Form I antigen. The great similarity in structure between the various forms implies that the Form II and III molecules are derived from Form I.

To determine whether the L3 antigen was previously described as an antigenic component of tumor types other than lung cancer, a computer-assisted literature search for released or secreted tumor-associated antigens was performed. We have reported several melanoma secretory protein antigens that are similar in size and carbohydrate composition to the L3 antigen (Natari et al. (1982) Cancerless. 42: 583-589; Bumor et al. (1982). Hybridoma 1: 283-292; and Wilson et al., (1981), Into Jay Cancer 28: 293-300 [Natali et al. (1982) Cancer Res. 42.
: 583-589; Bumol et al., (1982) Hybridoma 1: 283-29
2; and Wilson et al., (1981) Int. J. Cancer 28: 293-30.
I found 0]).

A small amount of one of the previously described monoclonal antibodies [465.12S; said Natali et al.]
First International Conference on Melanoma-Associated Antigens
It was available through attendees of the tional workshop on Melanoma Associated Antigens). We used this antibody in subsequent immunoprecipitation experiments to test for identity between the L3 antigen and the antigen recognized by antibody 465.12S. Both antibodies specifically precipitate Mr 94,000 protein from the culture medium of cells metabolically labeled with ³⁵ S-methionine. Depletion of the culture medium for either L3 or 465.12S antigen by immunoprecipitation results in a concomitant depletion of antigenic activity of the other antigen. This indicates that the antigenic determinants recognized by both antibodies are carried on the same molecule (Table 3). However, the epitopes recognized by the two antibodies are clearly different because no competition of antibody 465.12S for binding of ¹²⁵ I-L3 antibody could be detected.

Another antibody of the invention, designated L5, determines determinant sites on cell surface protein antigens unique to human NSCLC cells. The molecular weight of the protein, as determined by the above method and the ¹²⁵ I labeling method, was about
It is 135,000 Daltons. This antibody is of the Ig M class. The L5 antibody does not detectably bind to normal cells such as fibroblasts, endothelial cells and epithelial cells of major organs. The L5 antibody is produced from an L5 murine hybridoma. The L5 antigen is described in Cleveland et al., Journal, Of, Biological, Chemistry
No. 225, No. 1102-1106 (Cleveland et al. (1977) J. Biol.
Chem. 252: 1102-1106], the two characteristic Vs of Mr 24,000 and 16,000 are shown.
It has 8 protease fragments. Therefore, the L5 antigen is distinguished from other antigens of similar molecular weight.

Another monoclonal antibody of the present invention is L1.
Determining a determinant site on another cell surface protein antigen, designated 8 and unique to human NSCLC cells. The molecular weight of this protein is approximately 72,000 daltons as determined by both methods above. Antibody is of Ig G ₁ isotype. The L18 antibody binds to a small extent to normal cells, especially spleen cells. Also included within the scope of the invention are useful binding fragments of the above monoclonal antibodies, such as Fab, F (ab ') ₂ , Fv fragments. This antibody fragment can be obtained by a conventional method. For example,
Useful binding fragments are produced by peptidase degradation of antibodies with papain or pepsin. The term useful binding fragment means that the fragment competes with the antibody for binding sites on its cognate antigen.

The above specific examples of the novel antibodies of the invention are:
It is intended, but not meant to be limiting, for antibodies that bind to specific determinant sites on their respective antigens and are of a unique class and isotype from mouse sources. Antibodies having functional equivalence to the above antibodies, as well as the above antibodies from any of mouse sources, other mammalian sources, including humans, or other sources or combinations thereof are Other classes, such as Ig M, Ig A, IgE, etc., including isotypes within the class, can be used as well. The term "functional equivalence" means that the antibody is capable of binding to the determinant sites mentioned above and is capable of competing with a particular antibody of the invention for such sites. That is, such an antibody binds to such a determinant site when combined with a cell or cell fragment bearing such a determinant site or an analyte containing a secreted antigen, and It will inhibit the antibody of the invention from binding to the site.

One method of the invention involves measuring the presence of a malignant condition in lung tissue. The term "malignant condition" refers to the presence of dysplasti, neoplastic malignant or neoplastic cells, which also include carcinoma. The analyte is contacted or combined with the monoclonal antibody of the invention. Contacting is performed under conditions for binding of the antibody to malignant cells. After contact, the presence of antibody binding to malignant cells in the specimen is observed. That is, the specimen is examined for immune complexes between the antibody and the antigenic site. This immune complex formation is related to the presence of malignant cells in the specimen.

Particular examples of the method according to the invention, given by way of illustration and not limitation of the invention.
Is a method for detecting tumor cells in resected tissue. The above method is applied to a specimen which is a section of the tumor obtained after removal of the tumor. Excised tumor is processed to obtain sections, which involves first freezing the tumor or tissue, usually immediately after resection. The frozen layer of tissue is then cut into sections, for example using a cryostat.

Tumor sections obtained as described above are
It is contacted with a monoclonal antibody of the invention and then with a second antibody, directed against said monoclonal antibody, which is labeled with a detectable label. The excised specimen, eg a section of a tumor, is contacted with a first monoclonal antibody under conditions for the binding of the antibody to malignant cells. The incubation is carried out at about 20-3 in a suitable container such as a glass Petri dish.
It is usually carried out at a temperature of 0 ° C. for about 15 to 30 minutes in an aqueous medium such as phosphate buffered saline containing a small amount of sodium azide. The amount of antibody used is
It is usually sufficient to provide detectable binding, ie, provide a detectable number of immune complexes between the antibody and the determinant or antigenic site of interest.

Following incubation, the sections are washed to reduce or eliminate non-specifically bound antibody, and then by binding of the monoclonal antibody to cells of the specimen bearing the antigenic site. Interrogated to observe the complex. Binding is associated with the presence of malignant cells in the section. Thus, binding is measured, for example, by contacting the analyte with a labeled specific binding partner for the monoclonal antibody. The label is capable of emitting a detectable signal and may be a radioactive label, a chromophore such as a fluorophore, an enzyme or the like.

An example of a technique using the above approach is:
Immunofluorescence staining method. In this technique, frozen sections of tumor are fixed on glass slides with acetone and incubated with the monoclonal antibody, eg in Petri dishes. After washing with a suitable buffer such as, for example, phosphate buffered saline, the section is transferred to a Petri dish and can be, for example, a labeled antibody specific for the monoclonal antibody used. The antibody is contacted with a labeled specific binding partner for the antibody. In most cases, since the monoclonal antibody is of murine source, labeled anti-mouse immunoglobulin specific for the monoclonal antibody can be used. Such immunoglobulins can be produced by standard techniques by injecting a suitable host with mouse antibodies, allowing sufficient time, and harvesting anti-mouse immunoglobulin from the blood of the injected host.

After a second wash of the slide, for example with aqueous buffer, the sections are covered with a fluorescent antibody-containing fluid and a cover glass, and then to measure the binding of monoclonal antibodies to the sections. Was examined by fluorescence microscopy. Measuring binding also includes identifying the location of such binding in the analyte. Binding of the monoclonal antibody to the analyte can also be measured by using the monoclonal antibody covalently linked to a label capable of producing a detectable signal, such as a radioactive substance, a chromophore containing a dye and a fluorophore, or an enzyme. . The number of labels used per antibody is usually determined by the requirements of the diagnostic method in which the labeled antibody is used and the effectiveness of the sites for attaching the label to the antibody.

Methods for attaching labels to antibodies and antibody fragments are well known in the art.
Such a method is described in U.S. Patent Nos. 4,220,450 and 4,23.
Found in 5,869, 3,935,074 and 3,996,345. Yet another example of a technique in which the monoclonal antibody of the present invention is used is immunoperoxidase labeling (Galliguse et al., International Journal).
Ob Cancer (1982) 29: 511-515 as modified by Steron Berger, Immunocytochemistry, John Willie and Sons, New York, 1979, pp. 104-109 [Sternberger, Immunocytoc
hemistry, John Wiley & Sons, New York, 1979, pp104-169
as modified by Garrigues et al., Int.J.Cancer (198
2) 29: 511-515]). The tissue to be tested is fixed on a support such as a glass slide using a suitable solvent such as acetone. The tissue is then incubated with the monoclonal antibody and washed to remove unbound antibody. The tissue is then incubated with rabbit anti-mouse IgG, washed to remove unbound antibody, combined with mouse peroxidase-antiperoxidase complex and washed to remove unbound conjugate. , And then treated with a substrate for the enzyme. Following this processing, the slide was examined for a detectable signal.

The antibody of the present invention can be used in a method for measuring the presence of a malignant condition in a sample of exfoliative cells from the lung such as sputum. The term "exfoliative" refers to isolated cells or aggregates of cells obtained by rubbing or washing the surface of a tissue (these cells are removed individually or in scales or plates. ) Is meant to consist of. Exfoliative cell specimens should be distinguished from excised tissue, such as those obtained by biopsy. Contact between the analyte and the antibody is made under conditions for binding of the antibody to the antigenic site. After contact, the presence or absence of binding of the antibody to the antigenic site is measured and is related to the presence of a malignant condition in the lung.

To determine the presence of malignancy in the lung,
The sputum sample provides the exfoliative cell specimen used in this method. This method finds utility in detecting a malignant state in exfoliative cell specimens from the bronchus or the gastrointestinal tract including the pharyngeal mouth and mouth. The exfoliated cell specimen was then contacted with the aforementioned antibody under conditions for the binding of the antibody to a specific antigenic site of the specimen to form an antigen-antibody complex. This antigenic site may be present on cells or cell fragments in the specimen.
Generally, the specimen is mounted on a suitable support, such as a slide, usually made of glass or other suitable material. Exfoliative cell specimens are usually smeared onto the slide to give a thin layer of specimen on the surface of the slide. Contact between the antibody and analyte is usually performed in an aqueous buffered medium. The buffers used are phosphate, Tris,
Including bicarbonate and the like. The pH is related to the nature of the analyte and the antibody and is usually within the range of 5-8. Aqueous medium is about 0
It may further comprise an organic polar solvent in an amount of -40%. Organic polar solvents are water-soluble and usually have about 1-10 carbon atoms and about 1-4 oxygen atoms. The antibody is present in an aqueous medium at about 1-100 μg / ml, preferably about 1
It is present at a concentration of 0-20 μg / ml. The temperature during contact between the analyte and the antibody is about 4-40 ° C, preferably 10-30.
℃. The contact period is usually about 15 to 120 minutes, preferably about 30 to 60 minutes.

After contact between the analyte and the antibody, the support is usually treated to remove unreacted antibody. Usually, this is done by washing the support with an aqueous, usually buffered, medium. The presence of antibody bound to the antigenic site in the specimen, where this binding is associated with the presence of a malignant condition at that location, is then observed. That is, the specimen is examined to determine the number of antigen-antibody (immune) complexes formed. It should be noted that in some instances, a very small number of antigenic sites of interest may be found in exfoliative cell specimens. However, in malignant conditions, there are multiple antigenic sites, and this latter condition is measurable in the presence of many malignant states of the antigen-antibody complex, so this method Easy to identify from non-malignant condition. Means for emitting a detectable signal are incorporated into the assay system to provide a measure of the presence of binding. For example, the antibody used in the assay can be linked to a label capable of producing a detectable signal. Labels can be radioactive substances, chromophores including dyes and fluorophores, enzymes or the like. The number of labels used for an antibody is usually determined by the requirements of the method and the effectiveness of the sites for attaching the label to the antibody.

Alternatively, the washed slides can be contacted with a labeled specific binding partner for the antibody, which can be, for example, a labeled antibody specific for the antibody used. If the monoclonal antibody is derived from a mouse source, a labeled anti-mouse immunoglobulin specific for the antibody used in the method can be used. Such immunoglobulins can be produced by standard techniques by injecting a monoclonal antibody into a suitable host, at an appropriate time, and harvesting anti-mouse immunoglobulin from the blood of the injected host. If a labeled specific binding partner for the antibody is used, the slide must be washed again with aqueous medium before examining the slide for fluorescence.

To determine the presence of binding between the antibody and cell specimen when a fluorophore label is used, slides are examined for fluorescence, usually using a fluorescence microscope. If labels other than fluorophores are used, the slide or specimen is examined for the formation of precipitates, colors and the like. The above description has largely been devoted to the use of the antibodies of the invention in immunofluorescence techniques. However, the antibody of the present invention can be used in most assay methods including the antigen-antibody reaction. The test method group has homogeneity and heterogeneity.
ogeneous]. In the homogeneity assay method, the analyte can be a biological fluid such as serum, urine, and the like, or the analyte can be lysed and clarified to remove debris. For example, the antibody L3 has been used by the present inventors to measure the concentration of cognate antigen in serum from cancer patients, ie the type I, type II or type III L3 antigen, Cancer patients were found to have higher levels of this antigen compared to normal controls. Immunological reactions usually involve specific antibodies, labeled analytes and samples of interest.
The signal resulting from the label is altered, either directly or indirectly, upon binding of the antibody to the labeled analyte. Both the immunological reaction and the detection of its extent are carried out in a homogeneous solution. Immunochemical labels that can be used include free radicals, fluorescent dyes, enzymes, bacteriophages, coenzymes and the like.

In a heterogeneous assay method, the reagents are usually the analyte, the specific antibody, and the means for producing a detectable signal. The analyte is typically placed on a support such as a plate or slide and contacted with the antibody in the liquid phase. The support is then separated from the liquid phase,
Either the support phase or the liquid phase is examined for detectable signals using means for emitting such signals. This signal is related to the presence of the analyte in the analyte. Means for producing a detectable signal include the use of radioactive labels, fluorophores, enzymes and the like.
Examples of heterogeneous immunoassays include radiolabeled immunoassays (radioimmunoassays), immunofluorescence tests, enzyme-linked immunoassays, and the like.

For a detailed discussion of the above immunoassay techniques, see Edward T. Maggio.
(CRL Press Incorporated,
Boca Raton, Florida, 1980 [CRC Press Inc., Boca R
aton, Florida, 1980]) "Enzyme-Immunoassay". For example, U.S. Pat.Nos. 3,690,834, 3,791,932 and 3,817,837
No. 3, No. 3,850,578, No. 3,853,987, No. 3,867,517
No. 3,901,654, 3,935,074, 3,984,533
See also Nos. 3,996,345 and 4,098,876 (this list is not intended to be exhaustive).

The antibodies of the present invention are also described by Jay. Nakuru. Med. (1983) 24: 123-129 [J. Nucl. Med. (1983) 24: 123
-129] and in Journal of Clinical Investigation (1983) 72: 2101-2114 [J.Clin.In
vest. (1983) 72: 2101-2114] in a manner similar to that described for malignant myeloma, can be used to image metastatic deposits in human patients bearing NSCLC. The antibody or fragment thereof is radiolabeled and administered intravenously to the patient, which is then imaged using, for example, a gamma camera.

The present invention also includes a diagnostic kit for performing the above-described method. In one embodiment, the diagnostic kit is in a packaged combination.
It consists of (a) a more specifically defined monoclonal antibody and (b) a conjugate of a specific binding partner for the above monoclonal antibody and a label capable of producing a detectable signal. The reagent may also contain ancillary agents such as buffers and protein stabilizers such as, for example, polysaccharides, as necessary to perform such methods. The diagnostic kit may further include, if necessary, other components of the signal generating system in which the label is one component, an agent for reducing background interference in the test, a control reagent, and a test. It may include a device and the like. In other embodiments,
The diagnostic kit comprises a conjugate of the monoclonal antibody of the present invention and a label capable of emitting a detectable signal.
Supplementary agents such as those mentioned above can also be present.

The antibody of the present invention can be used therapeutically. Antibodies with unique biological properties are useful as direct therapeutic agents. Antibodies can also be conjugated to toxins to form immunotoxins and to radiopharmaceuticals or agents to radiopharmaceutically or pharmaceutically form. Methods for producing antibody immunotoxins or radiopharmaceuticals are well known (eg, Cancer Treatment Reports (1984) 68: 317-328 [Cancer Treatment Reports (198
4) 68: 317-328]).

Another therapeutic use of the monoclonal antibodies of the present invention is one of the monoclonal antibodies of the present invention as an immunogen.
Immunization of patients with anti-idiotypic antibodies produced by using Such immunizations can result in active anti-tumor activity (eg, Nepom et al., Proceedings of the National Academy of Sciences of the United State of America (1984) 81: 2864-2867 [Nepom et al.,
Proc.Natl.Sci.USA (1984) 81: 2864-2867]). In a similar approach, a patient can be immunized with a purified form of the antigen, a peptide fragment of the antigen, or a modified form of the antigen.

Particularly, in an interesting aspect of the present invention, the L3 antibody of the present invention can be used in combination with other antibodies. This combination is at least effective in detecting said type of lung cancer, in particular undifferentiated cell lung cancer. For example, (US Patent Application No. 6 filed November 2, 1984)
Monoclonal antibodies L3 and L18 (disclosed in 67,521) are combined in an effective amount, ie an amount which gives a detectable combination for all of said lung cancers.

Some of the monoclonal antibodies of the present invention also define determinant sites on antigens associated with breast cancer and other cancers such as epidermal cell carcinoma of the vulva. Therefore, the antibodies of the present invention may find use in diagnostic or therapeutic products directed against such cancers.

[0047]

The present invention is further described by the examples illustrated below. The numerous methods used are first described. The material culture medium used was that of Gibco. The fetal bovine serum used was that of Zivco or Hyclone. Teflon-coated multiple well microscope slides (12 wells) were from Melloy. Corning is a plastic culture dish
Or the one of Costar was used. The V8 protease and bovine serum albumin used were those of Sigma. Neuraminidase and Pansorbin [P
ansorbin] (staphylococcus with formalin fixed
S. aureus) is Calbiochem
I used the one. Endoglycosidase H is Miles
I used the one from [Miles]. Nitrocellulose (BA85,0.
45 μ) used from Schleicher and Schuell. Protein A-Sepharose, CNBr activated Sepharose 4B, Sephadex [G-10], DEAE
As Sephacel and Sepharose CL4B bound to protein A, those of Pharmacia were used. NP-40 used was Particle Data Limited [Particle Data Ltd.]. Polyethylene glycol, X-ray film and Kodavue-
The staining kit used was that of Kodak. Acrylamide and silver staining kits are available from BioRad
d] was used. Prestained molecular weight markers are available from Bethesda Research Laboratories, Bethe, Bethesda Research Laboratories, Bethesda, MD (BRL).
sda, Maryland (BRL)]. The 14 C-methylated molecular weight marker, 3 H-glucosamine, ³⁵ S-methionine and ¹²⁵ I used those of Amersham. The FITC-conjugated sheep anti-mouse immunoglobulin used was that of Tago. Paragon
n] Serum protein electrophoresis equipment is Beckman [Beckma
n] was used. Ampholin is El Cavy [LK
B] was used. Immunohistological Techniques Immunohistological studies on frozen sections include Immunochemistry, John Willie and Sons, New York, 1979, pp. 104-169 (Immunochemistry, John Wi.
ley & Sons, New York, 1979.pp104-169), as a correction by Garrigues et al. Jay. Cancer (1982) 29: 511-515 [Int.J.Cancer
(1982) 29: 511-515] was used. Target tissues for these studies were obtained surgically and frozen in isopentane pre-chilled in liquid nitrogen for 4 hours. Tissues were then stored in liquid nitrogen or at -70 ° C until use. Rabbit anti-mouse IgG [Sternberger-Meyer Immunochemicals, Inc. , Jaletswille, Maryland (S
ternberger-Meyer Immunochemicals, Inc., Jarettsvill
e, MD)] was used at a 1/50 dilution. Mouse peroxidase-antiperoxidase complex [PAP, Sternberger-Meyer Immunochemicals, I
nc)] contained 2 mg / ml of specially purified PAP and was used at a dilution of 1/80. Frozen sections were prepared, dried, treated with acetone and dried [Garrigues et al., 1982 (Garrigues et al, 198
2)]. The sections used for histological evaluation were stained with hematoxylin. To reduce non-specific background, sections were pre-incubated with 1/5 diluted normal human serum [Garigues et al., 1982 (Garrigues et al., 1982)]. Mouse antibodies, sheep anti-mouse IgG and mouse PAP were diluted in a solution of 10% normal human serum and 3% rabbit serum.

The staining method was as follows: serial sections were treated with specific or control antibody for 2.5 hours, incubated with rabbit anti-mouse IgG diluted 1/50 for 30 minutes,
3 to the mouse PAP complex diluted 1/80
It was due to exposure for 0 minutes. After each treatment with antibody, slides were washed twice with phosphate buffered saline (PBS). Immunohistochemical reactions were performed with freshly prepared 0.05% 3,3'-diaminobenzidine tetrahydrochloride [Sigma, St. Louis, MO (Siguma,
St. Louis, MO)] and 0.01% hydrogen peroxide in 0.05 M Tris buffer at pH 7.6 for 8 minutes. Furthermore, a 20 minute exposure to a 1% OsO ₄ solution in distilled water enhanced the dyeing.

Each slide was coded and the coded samples were checked by an independent laboratory. A typical slide was taken using a differential interferometric contrast optics [Zeiss-Monarski]. The degree of antibody staining is 0 (no reaction), +
(Most cells are not positive), ++ (at least one-third of the cells are positive), ++ (most cells are positive), ++++ (almost all cells are strongly positive) . Difference between + and 0 staining is ++
Since it is not as distinct as the difference between the + and + stainings, it was decided to count ++ or more as “positive” in the staining grade. Tumor cells and stromal cells were observed in tumor samples; staining was recorded for tumor cells as stromal cells did not stain at all or stained even weaker than tumor cells. Determining Antigen Location Subcellular localization of antigen was measured by measuring antibody binding to cells before or after permeabilization with nonionic detergents. Antibodies that bind to the cell surface of whole cultured cells are assayed by direct binding assay using ¹²⁵ I-labeled antibody (Brown et al., Proceeding of the National Academy of Sciences of the United States of America (1981).
78: 539-543 [Brown et al., Proc.Natl.Acad.Sci.U.
SA (1981) 78: 539-543]) or by indirect fluorescence using a (FACS) cell discriminator. Antibodies that bind to intracellular locations were measured by directly binding ¹²⁵ I-labeled antibody to cells, followed by immobilization with paraformaldehyde and subsequent permeabilization with the nonionic detergent NP-40. Was done. Binding Assay a) For the binding assay (Brown et al., Supra) performed by using a radiolabeled antibody, cultured cells (106) were heat-activated in culture medium (30 minutes at 56 ° C). ¹²⁵ I-labeled antibody in 100 μl of fetal bovine serum 10 ⁶ cp
m and incubated at 4 ° C for 30 minutes. PBS 5 ml
After the addition of cells, the cells were pelleted by centrifugation at 250 xg for 10 minutes. The supernatant liquid is sucked out,
The pellets were then counted for ¹²⁵ I. To measure non-specific binding, parallel incubations were performed with 10 μg of unlabeled antibody as competitor (Brown et al., Supra). In some cases, binding assays were performed on cell monolayers attached to plastic dishes in a similar fashion.

B) For binding assays performed on a FACSII cell sorter, cells were removed from their substrata using PBS containing 5 mM ethylenediaminetetraacetic acid (EDTA). Samples containing 1 × 10 ⁵ cells were first incubated with monoclonal antibody at a concentration of 2 μg / ml, followed by a 1: 200 dilution with fluorescein-conjugated goat anti-mouse antibody. The cells were then washed and resuspended in culture medium. Immediately prior to FACS analysis, propidium iodide was added to a final concentration of 1 μg / ml to stain non-viable cells. FACS
During the analysis, red-fluorescing cells were electronically displaced to examine only viable cells. The average intensity of fluorescein fluorescence was then measured for each antibody. Negative controls consisted of samples without monoclonal antibody, while positive controls consisted of monoclonal antibodies against HLA type 1 histocompatibility antigen. Staining was considered positive if the mean channel fluorescein was at least three times background. Protein Antigen Assay To identify protein antigens, lung carcinoma or fetal human lung cells were surface radioiodinated or metabolically labeled with ³⁵ S-methionine. Antigen was incubated with monoclonal antibody and goat anti-mouse Ig
Isolated from cell lysates by addition of G and adsorption to Staphylococcus aureus. Immunoprecipitates were washed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described above [Brown et al., Supra (Brown et al. Supra)]. Hybridoma cells producing specific antibodies labeled with ³⁵ S-methionine had 25 mM.
Contains Hepes buffer, 4 mM L-glutamine, 4.5 g / l glucose, 10 mM non-essential amino acids, 100 units / ml penicillin, 100 μg / ml streptomycin and 15% heat-inactivated neonatal calf serum (NCS) Microtiter wells were seeded in Dulbecco's minimal essential medium without methionine. The cells were mixed with 0.1 mCi ³⁵ S-methionine and 3
Contacted at 7 ° C. for 6 hours, and the supernatant was removed and centrifuged to separate the cells. Isotype determination a) Ouchterlony immunodiffusion method An aliquot of the supernatant of a particular hybridoma was placed in the central well of a 2% agar plate. Monospecific rabbit anti-mouse Ig isotype antibody (Meloy) was placed in the outer wells and the plates were incubated for 2 hours at room temperature and then overnight at 4 ° C.

B) 96-welp made of flexible polyvinyl chloride
Rate (Coaster) is 0.1 m for 2 hours at 37 ℃
coated with g / ml goat anti-mouse Ig antibody and at 37 ° C
It was countercoated with a 3% BSA solution for 2 hours. Hybrido
Ma supernatant was then incubated for 2 hours at 37 ° C.
After washing with PBS, play with bovine serum albumin (BSA)
The test was performed with a single feature that was bound to peroxidase at 37 ° C for 2 hours.
Heterosexual rabbit anti-mouse Ig isotype antibody (Zymd [Zym
ed]). After washing the plate
1 mg / ml orthofe in 0.1 M citrate buffer pH 4.5
Nylene diamine and 0.03% H₂O₂And incubate
Was cut. Dynatec ELIS with optical density at 630 nm
A plate reader [Dynatec ELISA plate reader]
Was measured.Staphylococcus aureus protein A binding assay Add microtiter wells with 5% NCS and PBS.
And 0.02% NaN₃Incubate with
I sucked it out. 25 μl of epidermal pore suspension (2 x 10 ⁷cell/
ml) to each well and add 25 μl of the specified
Incubated with antibody. Plate for 7 minutes at 1200 rpm
Centrifuge at 50% NCS / PBS / NaN₃so
Wash twice, 25 μl¹²⁵I-Staphylococcus aureus Pro
Thein A (about 50,000 cpm / 25 l) was added. That plate
Were incubated for 1 hour at 25 ° C and 5% NCS / PB
S / NaN₃It was washed twice with and dried. Cut the bottom of the well
Separated and measured with a gamma counter.Cell culture Cal-1 lung carcinoma cells
Obtained from the Kantai Culture Collection. Increase
Culture medium [50 units of penicillin per ml, per ml
50 μg streptomycin and 10% (v: v) w
Dulbecco's Modified Eagle Medium (D) containing fetal calf serum (FCS)
The cells were maintained as a medium monolayer culture. 0.05 cells
% Trypsin and 0.02% EDTA solution
Therefore, the blood count was measured.Indirect immunofluorescence test Cells were trypsinized and collected per well (6 mm diameter)
5-20 x 10³Teflon-coated multi-well slides at a density of
It was placed on (Meloy Labs). Before experiment start
The cells were kept at 37 ° C for about 18 hours. Remove medium and attach
The cells are treated with 0.5 mM CaCl₂And 0.5 mM MgCl₂Phosphorus containing
Acid buffer solution (PBS, NaCl, 0.14M; KCl, 3 mM; Na₂HPO_Four× 7H₂
O, 8 mM; and KH₂PO_Four, 15 mM) once. That
The cells were then incubated at 23 ° C for 20-30 minutes in a 25 microliter
Add Laformaldehyde solution (0.5% in PBS) and in situ
Fixed Wash cells and incubate intracellular antigen at 23 ° C for 5 minutes
Dew by adding PBS containing 0.2% (v: v) NP40
I let it out. After that, excess aldehyde groups are reduced at 23 ° C.
Binding buffer [50 mM BES (N, N-
Bis [2-hydroxyethyl] -2-aminoethane sulfone
Acid), 0.1% (w: v) BSA and 15% FCS in DMEM]
Blocked. 10 μg / ml of antibody in binding buffer
20 to 25 microliters per well
Added in quantity. Incubate slides at 4 ° C for 1 hour
And washed with binding buffer. Fluorescein iso
Thiocyanate-conjugated (FITC) secondary antibody (goat anti-mouse IgG
And IgM) were added at 4 ° C. for 1 hour. FITC used
Conjugate dilution is any signal to background ratio
Was determined experimentally to bring Incubation
Slides, wash the slides with binding buffer and water,
Air dried. A small volume of anti-fade solution [generator
Chic Systems Corporation (Genetic Sys
tens Corporation)] and cover glass
Leitz Orthoplan fluorescence spectroscopy microscope (Leitz Orthoplan
plan fluorescence microscope).
Examine the slide under a 40 × oil immersion objective and use Kodak
 I took a picture with Try X Pan film.Metabolic labeling A. ³⁵ S-methionine Cells were harvested by trypsinization. Trypan blue drainage
Viability, as measured by ablation, was usually greater than 95%. Four
× 10⁶Cells were harvested by centrifugation and the amino acid methionine
The cells were suspended in 1 ml of growth medium except for.³⁵S-Methio
Add nin (800 Ci / mmol) to a final concentration of 0.5 mCi / ml.
Well, incubate at 37 ° C for 1-6 hours with rotation
It was The labeled cells are then collected by centrifugation and
Washed before operation. In some cases, radiolabeled cell suspensions
Unlabeled methionine and 10% FCS in suspension
Dilute 10 times with growth medium containing and add to 100 mm tissue culture dish.
In addition, it was further incubated at 37 ° C. for 18 hours. That
Afterwards, adherent cells were washed with PBS before use. B. ³ H-glucosamine ³ Similar labels are used when labeling cells with H-glucosamine.
A ligation protocol was used. First, the internal glucose
Pool contains 10% dialyzed FCS but no glucose
Killed by incubating for 4 to 24 hours in a growth medium.
Thirsty Following a cell population showing> 90% viability after starvation
Used for the experiment. Cells are collected as above and at pH 7.4
10% dialyzed FCS, 0.01M HEPES (N-2-hydroxy
Ciethylpiperazine-N-2-ethanesulfonic acid) and 0.
5M Ci³Contains H-glucosamine [40 Ci / mmol]
Resuspended in 1 ml glucose-free growth medium. At 37 ° C
After labeling by spinning for 3 hours, cells are collected by centrifugation and PB
Washed with S.Iodination Greenwood et al. (1963) Biochemical Journal
89: 114〜123 (Greenwood, et al, (1963)Biochem, J. 89:
114-123)The chloramine T method is used as described by
Use the protein¹²⁵Radiolabeled with I. Tan
1 to 2 mCi of sample (50 μg)¹²⁵I (Ca
Diluted to 0.5 ml in PBS containing (unrear). 10μ
l of freshly made chloramine T solution (containing 10 μg)
Was added and reacted at 23 ° C. for 5 minutes. Reaction was 20 μg Na₂S
₂O_FiveAnd add a solution containing 3 μg of KI to finish.
It was Unreacted¹²⁵I containing 1 mg / ml bovine serum albumin
Sephadex G-10 equilibrated with PBS
-10) Removed by chromatography on a column.
Specific activity of the labeled monoclonal antibody used in this study
The sex is about 3 × 10 per nanomol.⁹It was cpm. Radiolabeling
Samples containing L3 antigen before treatment with Sephadex G-1
It was desalted with a 0 (Sephadex G-10) spin column.Immunoprecipitation analysis 1.Preparation of extract Collect metabolically labeled cells by centrifugation, 2-4 x 10 per ml
⁶Cell ratio NaCl, 0.15M; Na₂HPO_Four, 0.05
M; Sodium deoxycholate 1% (w: v); Triton X
-100 (Triton x-100), 1% (w: v) and SDS 0.1%
Solubilized in RIPA buffer containing (w: v). 4 ° C
After incubating for 10 minutes, centrifuge the mixture.
Clarification with a separator (147,000xg, Ti 70.1 rotor for 30 minutes)
Turned into Remove the supernatant and collect the radioactivity uptake in liquid scintillation.
It was measured by a ration counter. 2.Immunoprecipitation ³⁵ S-methionine and¹²⁵1-2 x 10 for I⁷cp
m (acid precipitation) or³4 × 10 for H-glucosamine
⁶4 aliquots of cell lysate containing cpm (acid-precipitating)
Incubate with 1 μg of antibody for 0.5-1 hours at ℃
It was When the labeled culture medium was analyzed, the total cell extract analyzed
Aliquots equivalent to the proportion of output were used. Affinite
Purified goat anti-mouse immunoglobulin (1-2 μg; goat)
Were prepared by immunizing mice with mouse immunoglobulin)
Add solution and incubate at 4 ° C for another 10-
It continued for 60 minutes. 50 μl formalin-immobilized staphylococcus
Add 10% solution of cas aureus (pansorbin)
Well, after incubating at 4 ℃ for 5 minutes, the immunoprecipitate was centrifuged.
Collected by separation (pansorbin 0.5% (v: v) NP before use
Wash once with 40 solution, once with 0.05% (viv) NP40 solution, and
Resuspend in the latter buffer containing mg / ml ovalbumin
Allowed). Precipitate the TNEN buffer (Trishydro)
Xymethylaminomethane, pH8.0, 0.02M; NaCl, 0.1
M; EDTA, 0.01M; and NP-40, 0.5% (w: v)) 3-4 times
Washed, frozen and stored at -20 ° C until use.Electrophoresis 1.SDS-polyacrylamide gel electrophoresis (SDS PAGE): Perform SDS PAGE according to above Laemmli
gave. The most frequently used separation gels are 10-20%
A linear acrylamide gradient and a stacking gel
The gel contained 5% acrylamide. Sample 5
Sample buffer containing 2% 2-mercaptoethanol
Boil in water and immobilize the immunosorbent before electrophoresis.
nt) was removed by centrifugation. The molecular weight standard is¹⁴C-methyl
Protein mixture (Amersham) or prestained sample (BR
L) was either. The amount of standard protein used
Myosin (200,000), phosphorylase b (92,40)
0), bovine serum albumin (BSA) (69,000), ovalbumin
(46,000), chymotrypsinogen (26,000), betalacto
Globulin (18,400), lysozyme (14,300) and sitok
It was Rom c (12,300). After electrophoresis, place the gel under vacuum
After drying, the position of the radiolabeled protein is determined by Kodak X-ray.
Determined by autoradiography using film. 2.Two-dimensional isoelectric focusing (IEF) -SDS PAGE ¹²⁵ Immunoprecipitates containing I-labeled L3 antigen as described
Prepared with 5% (v: v) 2-mercaptoethanol and
Resuspended in 50 μl of solution containing 1% (v: v) NP40.
Samples are heated at 95 ° C for 5 minutes and immunoadsorbent (immunoabs
orbent) was removed by centrifugation. Ampholin on sample
(ampholines) (pH 3.5-10; final concentration 4%) is mixed and solid
Urea was added to saturation. Journal of Offerings (1975)
Biology Chemical 250; 4007-4021 [O'Farrell (1
975)J. Biol. Chem.250; 4007-4021]
Electrophoresis and SDS PAGE were performed. Standard tongue
Pak quality marker (Bio-Rad Bio Rad) IEF dimension
Used to track the separation. The pH gradient obtained is
Determined from blank gels run in parallel. Gel 0.5c
sliced into m and sliced 1 ml of distilled H₂To O
After soaking for 1 hour, the pH of the resulting solution was determined. 3.Paragon electrophoresis: Producing serum protein electrophoresis
Paragon device (Beckma
Using a pre-formed agarose gel
went.Immunoblotting : SDS for analyzed protein
 Separation by either PAGE or Paragon
did. Bar the sample separated by SDS PAGE
Net (1981) Anal Biochemistry 112; 195 ~
203 [Burnette (1981)Anal.Biochem 112; 195-203]
Electrophoresis at 5V / cm for about 18 hours at 4 ℃
Transferred to nitrocellulose. Separated by Paragon
The sample is a sheet of nitrocellulose paper
By placing it on an electrophoretogram
I transferred it to nitrocellulose. Nitrocellulose,
Before use, transformer of the above Burnet without SDS
Hydrated in transfer buffer. Thickness
Place a layer of about 1 inch paper towel on nitrocellulose
And press down with a glass plate and reagent bottle. Trance
After transfer, judging by staining the gel, transfer
Completed within an hour. Blot the nitrocellulose blot.
Blotto, a milk-based cocktail
(John Johnson et al. Gene Analytical Technology
ー (1984)1: 3-8 (Johnsonet al.,Gene Anal. Technol
(1984)1: 3-8)) (1% (w: v) Carnation)
In milk powder and 0.2% (v: v) NP-40 in PBS)
Blocked by incubating for 1 hour. L3 antigen
1 to 4 x 10 in Blotto for 1 hour at 23 ° C⁶cp
m / ml¹²⁵Incubation of I-L3 antibody and blot
Clarified by Mr. Blot (Blott
Wash thoroughly with o) and dry before autoradiograph exposure.
It was

Binding of ¹²⁵ I-L3 antibody Cal-1 (Calu-1) cells (4-10 × 10 ⁴ ) were seeded in a 48-well cell culture dish 18 hours before the start of the experiment. Cells were washed with PBS and fixed with 0.5% paraformaldehyde solution in PBS for 20 minutes at 23 ° C. Wash monolayer with PBS, 5 at 23 ° C
Permeabilized by treatment with PBS containing 0.2% NP-40 for min. Various concentrations of ¹²⁵ I-labeled antibody at 23 ℃ for 60 minutes
Added in a final volume of 0.1 ml. The monolayer was then washed extensively with binding buffer and solubilized by the addition of 0.5M NaOH. Radioactivity bound to the cells was measured with a gamma counter. Samples were assayed in duplicate: Duplicate samples generally varied by 20% or less. In order to measure non-specific antibody binding, ¹²⁵ I-labeled antibody binding is usually
50-100 fold excess of unlabeled L3 that decreases by more than 50%
The same well was incubated with the antibody. Combined data is Scatchard (1949) Ann. N. Wai. Academy Science 51: 660-672 [Scatchard (1949) Ann.NYAcad .
Sci. 51: 660-672], the affinity constant (Ka) for the ¹²⁵ I-L3 antibody was determined to be about 1 × 10 ⁸ M ⁻¹ . ^{If the} cell monolayer was not treated with NP40 prior to the addition of ¹²⁵ I-L3 antibody, antibody binding was reduced more than 10-fold. Competitive Binding Assay L3 antigen activity was measured by determining the ability of the antigen to inhibit the binding of ¹²⁵ I-labeled L3 antibody to formalin-immobilized monolayers of Cal-1 cells. Cell monolayers were prepared as described above. ¹²⁵ I-L3 antibody was added to cells in the presence or absence of a concentration of 0.4 μg / ml of a solution containing L3 antigen activity. The final assay volume was 0.1 ml. Under these conditions, ¹²⁵ I-L3 antibody binding was> 95% specific, as judged by the ability of the unlabeled L3 antibody to compete for binding. Various dilutions of test solution were added and binding of ¹²⁵ I-labeled antibody was measured as described above. Typical inhibition curves for the various stages of purification of L3 antigen found in culture medium and in normal human serum are shown in Figure 1 (Supplementary Documents). One unit of L3 antigenic activity is defined as the amount required to inhibit the binding of ¹²⁵ I-L3 antibody added at 0.4 μg / ml by 50%. Immunoprecipitates of glycosidase treated ³⁵ S-methionine or ¹²⁵ I-labeled L3 antigen were prepared as described above. The pelleted immunoabsorbant was resuspended in 25 μl digestion buffer, 5 μl volume containing 5 milliunits of the appropriate enzyme was added and the sample was incubated at 37 ° C. for 18 hours. Concentrated SDS PAGE sample buffer was added and the sample was electrophoresed. The digestion buffer for neuraminidase treatment contained:
NaCl 0.15M; sodium acetate pH 5.3, 0.05M; Ca
Cl ₂ , 0.1% (w: v); and phenylmethylsulfonyl fluoride, 0.1% (w: v). Endoglycosidase H (E
For processing, the digestion buffer contained the following: Tris-HCl pH 6.5 ^, 0.025 M; SDS 0.2
% (W: v); and phenylmethylsulfonyl fluoride, 0.1% (W: V). Protein measurement Bradhold (1976) Anal Biochemistry using bovine serum albumin as a standard for protein concentration.
72: 248-254 [Bradford (1976) Anal.Biochem .72 : 248-25
It was measured by the method of [4]. Preparation of L3 Sepharose Lyophilized CNBr-activated-Sepharose
4B was reconstituted with 1 mM HCl for 30-60 minutes at 4 ° C. The resin was washed once with the same solution, collected by centrifugation and 1M
A solution of NaCl and 0.2 M sodium bicarbonate (pH)
8.3) in L3 antibody at 3.5 mg / ml. Mix 4
Incubated for 16 hours with rotation at ° C. The resin was collected by centrifugation and excess active groups were blocked by addition of 1-4 volumes of 0.25 M glycine solution at pH 8.3. The resin was then collected by centrifugation and washed several times with PBS before use. The coupling efficiency was assayed by measuring the absorbance at 280 nm of the antibody solution before and after coupling,
It was determined to be about 10 mg of L3 antibody per ml of packed resin. After gel staining SDS PAGE, the location of protein bands was determined by Coomassie blue staining or a commercial silver (BioRad Bio Rad) or nickel (Kodak) based staining kit. Determination of amino terminal sequence by Edman degradation. L3 antigen from culture medium was treated with 0.1 M Tris-HCl, p
Reduction with dithiothreitol (20 mM) in 0.1 ml of solution containing H 8.5 and 0.1% SDS (w: v) at 50 ° C. for 2 hours.
Then, the antigen was added to iodoacetamide (45 mM) for 30 minutes at 20 ° C.
Treated, S-carboxamide methylated and subjected to preparative SDS PAGE on a 10% acrylamide gel (15 mm thickness). After electrophoresis, the gel was loaded with Coomassie Brilliant Blue (0.5% by weight in 10% acetic acid and 30% isopropanol).
%) And decolorized with a solution of acetic acid (5% v: v) and methanol (17% v: v). The stained L3 antigen band is cut out with a razor blade and immediately ECU-040 Electroelutor / Concentrator
(C.B.S. Scientific Corporation San Diego California (CBS. Scient
ific Co., San Diego, Ca)) for electroelution and electrodialysis. Serum-derived L3 antigen was treated similarly, except that the sample was not carboxamide methylated. The total recovery of the method is S for analysis by silver staining.
Approximately 80 protein was calculated by DS-PAGE.
%Met. Automated Edman degradation with about 100 pmol S-
Using L3 antigen from conditioned medium carboxamide methylated (based on yield of identified Met-6) and unmodified L3 antigen from 38 pmol serum (based on yield of identified Val-1) Performed in a gas phase sequencer (Model 470A, Applied Biosystems, Incorporated, Foster City California). Phenylthiohydantoin amino acids were analyzed by reverse phase HPLC (detection unit, 2 pmol) using an IBM Instruments' Cyano column using a sodium acetate buffer / tetrahydrofuran / acetonitrile gradient for elution (Applied Biosystems). , Protein Sequence Users Bulletin NO.2, 1984). Example 1 Preparation of monoclonal antibody Monoclonal antibody for 3 months BALB / c mouse for 4
Produced by immunizing with human tissue from one of four different sources: (1) pleural exudate from a patient with metastatic non-small cell lung cancer, (2) cultured cells from non-small cell lung cancer, and (3) Lung tissue from 3-4 month old human embryos. Immunization was performed by injecting approximately 10 ⁷ cells into the abdominal cavity of the mouse 3 to 4 times. Three days after the final immunization, the splenocytes were removed, suspended in culture medium and fused with NS1 mouse myeloma cells (Koehler and Milstein, supra.
er and Milstein)). The mixture was inoculated to form a low density culture resulting from a single fused cell (clone); the technique used for hybridization was as described by Ye et al. Jay. Cancer (1979) 29 : 269-275 [Yeh, e
al ., Int . J. Cancer (1979) 29 : 269-275].

The supernatant from the hybrid cells was EL
ISA assay and autoradiographic indirect¹²⁵
I-labeled protein A assay [autoradiographic indi
rect ¹²⁵I-labelled prote in A assey] (Brown et al.,
Journal of Immunology Methods (1979)
31: 201〜209 [Brown et al., J. Immunol. Meth. (1979)31: 2
01-209]), and
To extracts from tumors used for immunization containing
Was screened. These extracts are
ー [Colcher et al.] (Cancer Research (1981
Year)42: 1451 ~ 1459 [Cancer Res., (1981)42: 1451-145
9]); adjusted using the method modified from Yey et al. (Supra).
Made For this, the tissue is washed with PBS and
For suspended and complete tumors, stainless steel sieve
It was done by squeezing through. After this,
1 mM phenylmethylsulfonyl fluoride (cal
Biochem-Bering Corporation Calf
San Diego, Orenia [Calbiochem-Behring Corp., Sa
n Diego, CA]) including 1 mM NaHCO₃Is added,
Next, add this substance to the Dounce homogenizer B
Homogenize on ice with 50 strokes of pestle
It was After centrifugation at 27,000 xg for 15 minutes, the supernatant is removed.
Discarded and pellet resuspended in PBS for 1 min
Sonicated and stored at -70 ° C.

Hybridomas that produced antibodies that bind to cell membrane extracts were cloned, grown in vitro and further tested for antibody specificity. This test was performed by using the immunohistological techniques described above to test the ability of the antibody to bind to frozen sections of lung cancer, other tumors and normal human tissue. These hybridomas that produce antibodies specific for human lung carcinoma were recloned, expanded, and injected into pristane-treated 3-month-old BALB / c mice, where they grew as ascites tumors. Let

The antibody secreted into ascites is protein A.
In Sepharose (Aye et al., Immunochemistry, (1978) 15 : 429 [Ey et al. Immunochemis.
try (1978) 15 : 429]) or Sephacryl S-300 [Seph
acryl S-300] and purified by gel filtration. Purified antibodies may be further subjected to additional specificity tests by immunohistology, whole cell binding assays to determine if the antibody has bound to the cell surface, and radioimmunoprecipitation tests as described above. Used for characterization.

Monoclonal antibodies L3, L5 and L18
Were produced from the corresponding hybridomas as described above. These antibodies exhibited the properties described above herein. L
The cell lines called 3, L5 and L18 were ATCC (American Type Culture Collection, 12301 Park Lawn Dahl.
ction, 12301Parklawn Dr., Rockville, Maryland 20852)
And have been assigned accession numbers HB8626, HB8627 and HB8628, respectively. The cell line L3 was re-deposited on October 25, 1984. Example 2 Purification of L3 antigen from serum-free conditioned medium Preparation of serum-free conditioned medium Cal-1 cells were placed in a rotating bottle (850 cm) in the presence of growth medium.
^The cells were grown to confluence in ² ). The monolayer was washed 3 times with 50 ml of serum-free growth medium and the second wash was left on the cells for 30 minutes at 37 ° C. The cells were then incubated in the presence of 150 ml serum free growth medium. After 3 days, collect the medium and centrifuge to remove floating cells,
It was stored frozen at -70 ° C until use. 2. Preparation of concentrate Serum-free medium (790 ml) was ultrafiltered (Amicon PM1O membrane.
Amicon.PM1O membrane) to a volume of 17 ml. The concentrate is transferred and the membrane is PBS (11 ml)
Once washed by. Wash solution and concentrate are combined,
Centrifuged at 147,000 xg for 30 minutes in a Ti 70 rotor (Beckman). The supernatant was found to contain most of the L3 antigen activity and is hereinafter referred to as the concentrate. 3. Immunoaffinity chromatography This concentrate was sent to a Sepharose C14B column (1.5 ml) to remove substances having an affinity for Sepharose. The column was then washed with 3 ml PBS. This flow-through was combined with the wash and added to a 1 ml fill volume of Sepharose 4B conjugated with L3 antibody (10 mg L3 / ml resin). The mixture was kept under rotation for 18 hours at 4 ° C and then poured into a column made using a hypodermic syringe. The flow-through was collected and the resin was PBS (10 ml), 5M NaCl (5 ml) and 0.02
It was washed with a solution of M ammonium acetate (10 ml). L3 antigen is a newly made triethylamine solution
It was eluted from the column by adding (75 mM).
Since L3 antigen activity was unstable to elevated pH, the pH of the effluent was adjusted by collecting fractions (0.5 ml) in tubes containing 0.1 ml of 2M ammonium acetate. Fractions containing L3 activity were identified by using a competitive binding assay, pooled and stored frozen at −20 ° C. The results are summarized in Table 1. Example 3 Purification of L3 antigen from serum Serum collection Blood was collected from tumor patients or normal individuals at room temperature for 10
Coagulated for ~ 90 minutes. Samples are then placed in a Sorvall Clinical centrifuge for 30 minutes to 5 hours 4-6 before being centrifuged at 1600 rpm for 10 minutes.
Stored at ° C. Serum was separated from blood clots, aliquoted and stored frozen at -70 ° C until use. L3 antigen activity levels were not significantly different in serum or plasma from the same individual. Thawed aliquots generally should not be refrozen, as the antigenic activity was found to be sensitive to repeated freezing and thawing. For purification of L3 antigenic activity, a 10 ml aliquot of freshly frozen serum was thawed and diluted before use.
Clarified by centrifugation (Ti 70.1 rotor) for 60 minutes at 7,000 xg. 2. DEAE Sephacel chromatography 10 ml column of DEAE Sephacel was injected and PE buffer (sodium phosphate 0.02M. PH 7.12, 0.005M EDT
Equilibrated by A). The clarified serum was diluted with PE buffer to 130 ml before being applied to the ion exchange column.
Diluted to a volume of. 0.5 between sample application and elution
A flow rate of ml / min was maintained. The column flow-through contained 54% of the original serum protein (determined by absorbance at 280 nm) and had no detectable L3 antigen activity. The column was then washed with 10 column volumes of PE buffer. Elution of L3 antigen activity was achieved using a 50 ml gradient of 0-0.5 MNaCl in PE buffer. During the elution, 1 ml fractions were collected. After applying this gradient, the column was loaded with 0.5 M Na in PE buffer.
Washed with Cl and finally in the same buffer
It was washed with 1.5 M NaCl. Fractions containing L3 antigen activity were identified using a competitive inhibition assay and pooled. L3 antigenic activity was slightly tailed after elution of large amounts of serum proteins.

In some experiments, L3 antigen activity was partially purified by a slightly different procedure prior to radiolabeling and immunoprecipitation analysis. Serum proteins were first precipitated with 30% ammonium sulfate. This precipitate is H ₂ O
It was dissolved in and desalted (with Sephadex G-25) before being applied to the column of DEAE Sephacel. Elution of L3 activity was achieved by stepwise addition of NaCl solution. Fractions eluting between 0.25 M and 0.5 M NaCl were enriched for L3 antigen activity, followed by L
Used for radiolabelling of 3. Antigens prepared in this manner were indistinguishable by protease fingerprinting from radiolabeled antigen precipitated from preparations purified 3,000-fold or more. 3. Immunoaffinity chromatography The DEAE Sephacel column (14 ml) pooled fractions were applied to a Sephacel CL4B 8.4 ml column which was washed with 17 ml PBS. The washings were then combined with the flow through and the mixture was spun at 4 ° C. with Sepharose conjugated with antibody L3 (10 mg L3 / ml resin) with a loading volume of 1.5 ml.
Time equilibrated. This mixture was poured into a column,
The resin was then PBS (42 ml), PBS containing 0.2% (v: v) NP40 (28 ml).
ml), PBS (14 ml), 0.02 M ammonium acetate (28 m
l), 5M NaCl (28 ml) and finally 0.02 M ammonium acetate (5 ml). The L3 antigen was eluted by adding 8.4 ml 0.075M triethylamine as described above. The results are summarized in Tables 2 and 3. Example 4 Competitive Binding of L3 Antibody and Antibody 465.12S Unlabeled Antibody 465.12S or Unlabeled L
3 antibody is ¹²⁵ I-L3 antibody (final concentration 0.35 μg / ml)
Formalin-fixed H125 lung cancer cells mixed with
(50,000 / well). Unlabeled antibody was used as the hybridoma supernatant and each antibody was diluted to half its original volume in the final assay. The solution of antibody 465.12S used was sufficient to precipitate many antigens of the same valency as those precipitated from 1 microgram of purified L3 antibody from ³⁵ S-methionine labeled cell extract. Was included. The results shown in Table 4 show that antibody 465.12S does not compete for binding of ¹²⁵ I-L3 antibody. Thus, the two antibodies recognize distinct epitopes and are thus distinct antibodies and not functional equivalents. Example 5 Cleavage Fragment of L5 Antigen CALU-1 human lung cancer cells are described by Brown et al.
l.] (Journal of Immunology [J.Immunol.] No. 12
7, 539-546 (1981)) and surface labeled using lactoperoxidase catalyzed iodination. L5 antigen, which is the antigen defined by antibody L5, was precipitated from cell extracts and analyzed by SDS-PAGE performed under reducing conditions. When analyzed accordingly, the L5 antigen migrated as a single polypeptide chain of Mr 135,000. The gel region containing the L5 antigen was excised and a specified amount of Staphylococcus aureus [Staphyl
ococcus aureus] V8 protease and treated as described by Kleberand et al. [Cleveland et al.] above.
It was analyzed again by DS-PAGE. The resulting "fingerprint" of the cell surface iodinated V8 cleavage fragment is diagnostic of the L5 antigen. That is, the cleavage fragments of Mr 24,000 and 16,000 are characteristic of the L5 antigen and will distinguish it from other antigens of similar molecular weight.

[0058]

[0059]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12P 21/08 8214-4B G01N 33/574 A 8310-2J 33/577 B 8310-2J (C12P 21 / 02 C12R 1:91) (C12P 21/08 C12R 1:91) (72) Inventor Helstome, Carl Eric Washington, USA 98121 Seattle North East Server Drive 3925 (72) Inventor Horn, Diane Washington, USA 98121 Seattle West Olympic Place Number 404 202 (72) Inventor Linsley, Peter Washington, USA 98121 Seattle 9th West 2430

Claims (4)

[Claims] 1. An antigenic composition purified from a culture medium of human non-small cell lung cancer cells containing an unmodified, glycosylated form I L3 protein, the protein having the following N-terminal amino acid sequence: : 1 5 10 15 20 25 30 VNDGDMRLADGGATNQGRVEI-FYRGQWGTV having a molecular weight of about 94,000 daltons as measured by SDS polyacrylamide gel electrophoresis under reducing conditions and having a terminal residue of sialic acid residue N An antigen composition, characterized in that it has attached glycoside residues. 2. The composition of claim 1, further comprising a Form II L3 protein having a molecular weight of about 76,000 daltons as determined by SDS polyacrylamide gel electrophoresis under reducing conditions. 3. An antigenic composition purified from normal human serum containing an undenatured, glycosylated Form I L3 protein, wherein the protein has the following N-terminal amino acid sequence: 1 5 10 15 20. 25 30 VNDGDMRLADGGATNQGRVEI-FYRGQWGTV having a molecular weight of about 94,000 daltons as measured by SDS polyacrylamide gel electrophoresis under reducing conditions and having an N-linked glycoside residue whose terminal residue is a sialic acid residue An antigen composition comprising: 4. The composition of claim 3, further comprising a Form II L3 protein having a molecular weight of about 76,000 daltons as determined by SDS polyacrylamide gel electrophoresis under reducing conditions.